Book of Abstracts - Geyseco

FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology
P05-045: DEVELOPMENT OF STS MARKERS FOR
ANALYSIS OF GENETIC DIVERSITY USING EEG LI-
BRARY IN SOYBEAN
Seo, M.* – Lee, S.K. – Kim, Y.H. – Jeong, K.H. – Hwang, T.Y. –
Choi, M.S. – Yun, H.T.
National Institute of Crop Science, RDA
*Corresponding author e-mail: mjseo77@korea.kr
Several molecular markers are available for identifying germplasms
and for analyzing their genetic diversity. Some molecular
markers like RAPD and AFLP are undefined elements, whereas
SSR and STS markers are derived from defined elements. Especially,
sequence tagged site (STS) marker as a source of locus-
specific codominant marker is distinctive in that it is combined
advantages of PCR and RFLP. We performed this study to
develop the gene based DNA library and STS marker derived
from gene rich region for identifying of soybean cultivar. To develop
the euchromatin enriched genomic DNA (EEG) library of
soybean, we used DH5 alpha bacteria cell with the Mcr A and
Mcr BC system. One thousand four hundred forty EEG colonies
have been constructed in total. We analyzed blast search of NCBI
and Phytozome for the genetic information of sequenced colonies.
More than one hundred forty STS primer sets have been designed
based on the sequencing data of selected colonies. The developed
primer sets were applied for Hwanggeumkong to select
promising primer sets and more than one hundred Korean cultivars
were tested to analyze genetic diversity with selected primer
sets. Twelve primer sets were polymorphic in Korean cultivars
using five restriction enzymes. It is thought that these primers
could be useful for specific allele tagging in diverse germplasms
and for the functional study of soybean. (This work was supported
by grants from the R&D project (#200901FHT020609503) of
the National Institute of Crop Science of the Rural Development
Administration)
P05-046: THREE ENDO-ß-MANNANASE GENES EX-
PRESSED IN THE RADICLE TIP AND MICROPYLAR
ENDOSPERM PRIOR TO RADICLE EMERGENCE IN-
FLUENCE GERMINATION OF ARABIDOPSIS THALIA-
NA SEEDS
Iglesias-Fernández, R.¹* - Rodríguez-Gacio, M.C.² - Barrero-
Sicilia, C.¹ - Carbonero, P.¹ - Matilla, A.²
¹Universidad Politécnica de Madrid
²Universidad de Santiago de Compostela
*Corresponding author e-mail: raquel.iglesias@upm.es
Mannans are hemicellulosic polysaccharides in the plant primary
cell wall (CW). Mature seeds, specially their endosperm cells,
have CW rich in mannan-base polymers that confer a strong
mechanical resistance for the embryo protrusion. The rupture of
the seed coat and endosperm are two sequential events during
the germination of Arabidopsis thaliana. Endo-β–mannanases
(MAN; EC. 3.2.1.78) are hydrolytic enzymes that catalyze cleavage
of β1→4 bonds in the mannan-polymer. In the genome
of Arabidopsis, the endo-beta-mannanase (MAN) family is
represented by eight members. We have systematically explored
the expression of the eight MAN genes in different organs of
this plant and found that only four of them (AtMAN7, AtMAN6,
AtMAN2 and AtMAN5) are induced upon seed germination. Moreover,
in situ hybridization analysis shows that their transcript
accumulation is restricted to the radicle and to the micropylar
endosperm before protrusion, and this expression disappears
soon after radicle emergence. T-DNA insertion mutants in these
genes (K.O. MAN7, K.O. MAN6, K.O. MAN5), except that corresponding
to AtMAN2 (K.O. MAN2) that is weakly expressed
in germinating seeds, show a germination rate slower than that
of the wild type (Wt). K.O.MAN6 is the most affected in the
germination time course with a t 50
=48h, almost double than that
of the Wt (t 50
=25h). Our data demonstrate that AtMAN6, and at
a lower degree AtMAN7 and AtMAN5, are crucial players in the
germination process of A. thaliana seeds, possibly by facilitating
the endosperm rupture by the emerging radicle.
P05-047: ANALYSIS OF FIVE NOVEL CONSTITUTIVE
GENE PROMOTERS IN TRANSGENIC RICE PLANTS
Kim, J.¹* - Park, S.H.¹ - Yi, N.¹ - Kim, Y.S.¹ - Bang, S.W.¹ - Choi,
Y.D.²
¹Myongji University
²Seoul National University
*Corresponding author e-mail: jukon306@gmail.com
Novel constitutive gene promoters are essential components of
crop biotechnology. We report our analysis of five such promoters,
APX, SCP1, PGD1, R1G1B and EIF5, in transgenic rice
plants. The five promoter regions were linked to the gfp reporter
gene and transformed into rice. Using fluorescent microscopy
and qPCR, promoter activities were analyzed in comparison with
OsCc1, Act1 and ZmUbi1, previously characterized strong constitutive
promoters. The APX and PGD1 promoters direct high levels
of gene expression in all tissues and stages, producing GFP
at levels up to 1.3% of the total soluble protein. PGD1 is particularly
active in flowers and mature roots. The R1G1B is active in
the whole grain including the embryo, endosperm and aleurone
layer, and thus represents a constitutive promoter with activity
in whole seeds that has not been described previously. The
ZmUbi1 and R1G1B promoters are markedly less active in young
roots and mature leaves whilst the APX, PGD1, OsCc1 and Act1
promoters are highly active in both vegetative and reproductive
tissues. Overall, our results demonstrate that APX, PGD1 and
R1G1B are novel constitutive gene promoters that are highly active
at all stages of plant growth with distinct levels of activity.
P05-048: INHIBITION OF ENDOGENOUS PROTEASES
IN NICOTIANA TABACUM CV BRIGHT YELLOW 2 (BY-
2) SUSPENSION CELLS IMPROVES RECOMBINANT
PROTEIN PRODUCTION
Mandal, M. 1 * - Mandal, M.K. 2 - Fischer, R. 2 - Schillberg, S. 1 -
Schiermeyer, A. 1
1
Fraunhofer Institute for Molecular Biology and Applied Ecology
(IME), Department Plant Biotechnology
2
Rwth Aachen University Institute for Molecular Biotechnology
*Corresponding author e-mail: mkmiit2002@gmail.com
Recombinant proteins secreted to the plant suspension culture
medium are often degraded by endogenous plant proteases
resulting in low yields. Recently we have cloned different protease
cDNAs from BY-2 cells (NtAspP, NtCysP, NtMMP1 and
NtSerP). We have exploited the sequence information to generate
protease deficient tobacco BY-2 cell lines through the simultaneous
expression of antisense-RNAs against these endogenous
proteases. All established antisense cell lines showed reduced levels
of endogenous protease expression and activity at late stages
of the cultivation cycle. Strikingly, knockdown of the endogenous
proteases led to an elongated cell shape indicating involvement
of proteases in cell growth and development.
One of the antisense cell lines showing reduced proteolytic activity
in the culture medium was selected for the expression of the
recombinant full-length antibody 2F5 recognizing the gp41 surface
protein of HIV-1. This cell line showed significantly reduced
degradation of the 2F5 heavy chain resulting in four fold higher
accumulation of the intact antibody heavy chain when compared
to transformed wild type cells expressing the same antibody.
These data provide a basis for further improvement of plant cells
for the production of recombinant proteins.
P05-049: REGULATION OF XYLEM DEVELOPMENT IN
BRACHYPODIUM DISTACHYON
Ruth Valdivia, E. – Gianzo, C. – Fidalgo, J. – Cortiñas, L. – Freijeiro,
S. – Herrera, MT. – Revilla, G. – Zarra, I.* – Sampedro, J.
Universidad de Santiago de Compostela